The hematoxylin and eosin (“H and E”) staining technique is the most commonly used histological technique for the visualization of pathology in tissue specimens. A typical H and E staining system is composed of solutions comprising aluminum based hematoxylin, eosin, a differentiating solution, and a bluing agent. Hematoxylin is a natural dye that when complexed with aluminum ions produces a positively charged molecule that binds to deoxyribonucleic acid to produce a purple coloration of cell nuclei. Differentiation solutions typically are lightly acidic solutions that sharpen the contrast of the stained slide by removing excessive background staining from the tissue and slide. Bluing agents are solutions of a basic pH that when applied to stained specimens modify the structure of the bound hematoxylin to produce a vibrant blue/purple coloration. Eosin is a negatively charged dye that binds to positively charged proteins within the cytoplasm and connective tissue to produce a pink/orange coloration that contrasts with the blue/purple coloration of the nuclei.
H and E staining is most commonly performed in a “batch” format wherein automated stainers typically contain a series of staining reagent containers and utilize a robotic arm for the sequential movement of slides/racks from one container to another. Typically, a rack or holder is capable of carrying 20 to 30 slides although some staining instruments incorporate racks that hold fewer or greater numbers of slides per rack and in the case of at least one stainer, multiple racks may be places into a staining vessel simultaneously. Software controlling an automated stainer allows the operator to program the duration of each of the steps as well as the sequence of the steps. While the batch format of staining is simple, economical, and relatively rapid, there are drawbacks with the technique.
The movement of racks carrying slides from one container to the next results in the carry-over of staining reagents or water. Carry-over occurs due to the adherence of staining reagents or water to the slides, samples, the slide rack, etc. Carry-over is undesirable because it may affect the functionality of the downstream staining reagents in several ways. Carry-over dilutes the concentration of the dyes or functional reagents within a solution. Carry-over and dilution can also affect the functionality of the staining reagent by changing the characteristics of the solvent and thus affecting such parameters such as pH and ionic strength of the solvent. Finally, carry-over may introduce contaminants to the staining reagent. Acting through these mechanisms, carry-over negatively impacts the performance, functionality, stability, predictability, and capacity of the staining system. Although a single instance of carry-over may result in such negative impacts, the negative impacts of carry-over are especially problematic over the course of multiple staining runs as the negative impacts build or are amplified as the result of the cumulative effects of repeated carry-over.
Because of the negative effects of carry-over, the capacity or useful life of reagents in current staining systems is highly unpredictable. Reagents are often changed after a predetermined amount of use based on past experience or changed once they start to exhibit signs of or reach the point of failure. Therefore, solutions in current systems are often changed at different rates, i.e., at different times. This adds complexity and inefficiency as staining is continually stopped to change just one or less than all of the solutions. On the other hand, if all of the solutions are changed at once, some of them are discarded while still having useful capacity to process additional slides, and thus money is wasted.
Typical attempts to address the negative consequences of carry-over have involved replacing staining reagents with fresh solutions, either on a frequent regular basis or even for each individual sample. For example as described in U.S. Pat. Nos. 7,468,161 and 8,048,373. In addition, there have been improvements in individual reagent chemistries (hematoxylin, eosin, differentiator and bluing buffer) that may delayed some of the effects of carryover. In at least one case, there has been an attempt to eliminate the effects of carryover due to the mechanical design of the instrument (Dako CoverStainer). However, unpredictability and loss of staining effectiveness due to carryover still remains a major unresolved problem.